A reliable radiochromatographic assay technique for hepatic microsomal 16 α-hydroxylase activity towards oestrone 3-sulphate. Comparison between pigmented and non-pigmented mature guinea pigs

Abstract

A reliable procedure for the assay of liver microsomal 16 alpha-hydroxylation of oestrone 3-sulphate has been developed for the guinea pig. It is based on the rapid, quantitative separation of oestradiol and oestriol by Sephadex LH-20 columns after the chemical reduction and enzymic hydrolysis of the incubation products. Microsomal preparations and incubation conditions that optimized 16 alpha-hydroxylation of oestrone 3-sulphate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulphate were minimized. Conditions were established that yielded reaction linearity with respect to time and microsomal concentration. This hydroxylation had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Km values for oestrone 3-sulphate and NADPH, under the conditions used, were 14 microM and 0.17 mM respectively. 16 alpha-Hydroxylase activity was present in the liver microsomal fraction from heavily pigmented, female English Shorthaired guinea pigs. Much lower activity was detected in mature pigmented males and albino females. No activity could be demonstrated in mature, albino males.